CN114672586B - SNP molecular marker related to width character of papaya fruit, amplification primer, detection kit and application thereof - Google Patents
SNP molecular marker related to width character of papaya fruit, amplification primer, detection kit and application thereof Download PDFInfo
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Abstract
The invention provides an SNP molecular marker related to width traits of papaya fruits, an amplification primer, a detection kit and application thereof, and belongs to the technical field of molecular biology. The invention obtains an SNP molecular marker related to the papaya fruit width based on GWAS analysis and screening, wherein the SNP molecular marker is positioned at the 26693188 th basic group (Cpa 03g017160 gene CDs region) of a Chr03 chromosome and is named as Cpa03g017160:6929, and the SNP molecular marker is expressed in SEQ ID NO:1, wherein the G allele is significantly associated with papaya fruit width. The SNP molecular marker can be applied to detecting the high fruit width breeding material of the papaya, is beneficial to quickly detecting the high fruit width breeding material, assists in cross breeding, shortens the breeding period of new varieties, and has the advantages of low detection cost, no environmental limitation, high detection result accuracy and easy repetition.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SNP molecular marker related to width traits of papaya fruits, an amplification primer, a detection kit and application thereof.
Background
Papaya is a tropical evergreen fruit tree, a perennial fleshy herbaceous plant of the family Caricaceae, the genus Caricaceae, also known as Ninggua, Shigua, papaw, papaya, Mucuna Benincasa, papaya, Nelumbo nucifera. The method is originally produced in Mexico and Central America, is widely cultivated in tropical and subtropical regions all over the world, is introduced into China from the 17 th century, and is mainly produced in Guangdong, Hainan, Guangxi, Yunnan, Taiwan, Fujian, Sichuan Wenchang and Jiangxi. The fresh papaya has beautiful appearance, thin skin and thick meat, multiple tastes and sweetness, fragrant and sweet smell and rich nutrition. The ripe papaya flesh is yellow or red, and the carotene and lycopene are rich, so that the papaya flesh has excellent health-care effect and important edible value and industrial value. The development of papaya production greatly stimulates the development of related industries such as food processing, medicine and health, beauty and health care, breeding and the like, and has important significance.
The shape of fruit is an important factor influencing the yield of the papaya fruits, and the width of the fruits is an important factor influencing the weight of the single fruits and even the total yield of the fruits, so breeders often seek to breed varieties with larger width of the papaya fruits.
Single Nucleotide Polymorphism (SNP) mainly refers to a DNA sequence polymorphism caused by variation of a single nucleotide at a genome level. Compared with molecular markers such as early RAPD, AFLP and SSR, the SNP molecular markers have the advantages of wide distribution and more quantity on individual genomes, easiness in genotyping (polymorphism of SNP), suitability for rapid and large-scale screening and the like. However, at present, there is no report on molecular markers related to the fruit width trait of papaya, which is undoubtedly not beneficial to the breeding of excellent varieties of papaya.
Disclosure of Invention
In view of the above, the invention aims to provide an SNP molecular marker which is significantly related to the width character of papaya fruits, can be used for detecting or predicting the width of papaya fruits, and provides a basis for breeding excellent and high-yield varieties of papaya.
The invention provides an SNP molecular marker related to the width character of papaya fruits, wherein the SNP molecular marker has an A/G polymorphic site at position 197 on the basis of a sequence with a nucleotide sequence shown as SEQ ID NO. 1.
The invention provides a primer for detecting the width of papaya fruits based on the SNP molecular marker, which comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 2 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3.
The invention provides a kit for detecting the width of papaya fruits, which comprises the primer and a detection reagent.
Preferably, the detection reagent comprises an amplification buffer and a standard substance;
the standard substance is a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1.
The invention provides application of the SNP molecular marker, the primer or the kit in detecting or predicting the width of papaya fruits or papaya breeding.
Preferably, the papaya fruit genotype is GG type, which indicates that the papaya fruit has a wider trait;
papaya material with the genotype of GG is selected as follow-up breeding material.
The invention provides a method for breeding a wide-fruit papaya variety in an auxiliary manner based on the SNP molecular marker, the primer or the kit, which comprises the following steps:
1) extracting the genome DNA of the material to be selected;
2) taking the genomic DNA obtained in the step 1) as a template, and carrying out PCR amplification by using the primer to obtain a PCR amplification product;
3) sequencing the PCR amplification product in the step 2), determining the genotype of the 197 th base of the sequencing sequence, and when the genotype is GG, indicating that the papaya fruits of the material to be bred have the character of wide fruit shape, and using the papaya fruits as breeding materials for subsequent cross breeding.
Preferably, the reaction system for PCR amplification in step 2):
2× Rapid Taq Master Mix 12.5μl
10 μ M Forward primer 1 μ l
Mu.l of 10. mu.M reverse primer
ddH 2 O 9.5μl。
Preferably, the reaction procedure of the PCR amplification in the step 2) is 95 ℃ for 5 min; 30s at 95 ℃, 30s at 54 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃.
Preferably, after the PCR amplification product in step 2) is purified, the 497 bp-long amplification fragment is collected for sequencing.
The SNP molecular marker related to the width of the papaya fruit is positioned on a nucleotide sequence shown in SEQ ID No.1, and the 197 th base from the 5' end of the sequence is an SNP locus; the SNP locus is at the 26693188 th base of the chromosome of Chr 03. Cpa03G017160 gene related to papaya fruit width and obtained by screening the SNP molecular marker based on a GWAS analysis method is very significantly related (P is less than 0.01), has A/G polymorphism, and is very significantly related to papaya fruit width through experimental verification.
The method for breeding the wide-fruit-shape papaya variety with the assistance of the SNP molecular marker, provided by the invention, comprises the steps of taking a nucleotide sequence containing the SNP molecular marker as a template, designing a primer pair, carrying out PCR amplification by taking papaya genomic DNA as the template to obtain an amplification product with the length of 497bp, and sequencing to obtain the genotype of a material to be bred, wherein the genotype is GG, so that the material has the wide fruit shape property. The method provided by the invention is beneficial to quickly detecting the papaya breeding material with high fruit width, assisting in cross breeding, shortening the breeding period of new varieties, and has the advantages of low detection cost, no environmental limitation, high detection result accuracy and easiness in repetition.
Drawings
FIG. 1 is a Manhattan plot of SNP markers of the present invention that are significantly related to papaya fruit width;
FIG. 2 is a Manhattan diagram of a Chr03 chromosome local interval where SNP markers which are extremely significantly related to the width of papaya fruits are located and a linkage disequilibrium haplotype block diagram, wherein deepening markers in the interval are molecular markers screened by the invention;
FIG. 3 is a comparison of fruit width differences between different genotypes of SNP markers of the invention that are significantly associated with papaya fruit width, with the G allele being significantly associated with high fruit width.
Detailed Description
The invention provides an SNP molecular marker related to the width character of papaya fruits, wherein the SNP molecular marker has a nucleotide sequence shown as SEQ ID NO:1 (TGCTACTTCAG)
AAACATTAGAAAAAGAAGATCATACACATTAGAGGAGAATTTGTTTCTCTTTTTTGACAGTTTATATTTTCGGTTTGTTCTTTTGTTTTTTTTTACTTCATTTTGCTCTCACAATTACTATCAGATACTGGAATGTCGACTTGTGGGAAAATCTCTTCACAAAGCTGCTTAACAGTAGCCATGGAR（A/G）ATGATGACACAAAATTGCTGCGGAGTTTGAG
GGAGTCCTTTCAGTGGTACATCCATGATAACCCTGAGCTCTTAAAGAAACTAAAGCAATTATTGGCAAAGCAAAGGGCGTCTTTGTGTTCTTCTTAGTGTATATTATATCATTTCTTCAGAATCCACCCTAAAAGTGAATATAAATAATTTTAGGAAGTAAATATTTTTTTTTTGAAATCAGTCTGTTGTCCAAAAGGAACTTTGTGCGCACTAGTCATTCTGGCAATGTATTTACTTAATGTCTCCTTAGATAAAGAAAGATGTCTCC) is added into the sequence of the gene, and an A/G polymorphic site exists at the 197 th position on the basis of the sequence shown in the sequence.
The invention provides a primer for detecting the width of papaya fruits based on the SNP molecular marker, which comprises a nucleotide sequence shown as SEQ ID NO:2 (TGCTACTTCAGAAACAT)
TAG) and the nucleotide sequence of the forward primer is shown as SEQ ID NO. 3 (GGAGACATC)
TTTCTTTATCTA) is used. The source of the primer in the present invention is not particularly limited, and any primer known in the art may be used. In the examples of the present invention, the primers were synthesized by Beijing Oakuo Dingsheng Biotech Co.
The invention provides a kit for detecting the width of papaya fruits, which comprises the primer and a detection reagent.
In the present invention, the detection reagent preferably includes an amplification buffer and a standard. The amplification buffer solution is preferably PCR amplification MIX solution. The source of the amplification buffer is not particularly limited in the present invention, and any amplification buffer known in the art may be used. In the present examples, the amplification buffer was purchased from Novozian Biotechnology, Inc. The standard substance is a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1. The standard is used as a Marker for indicating a target amplification band.
Based on the fact that the SNP molecular marker is extremely obviously related to the width of the papaya fruit, the invention provides the application of the SNP molecular marker, the primer or the kit in detecting or predicting the width of the papaya fruit or in papaya breeding. The genotype of the papaya fruit is GG type, which indicates that the papaya fruit has wider characters. And selecting papaya materials with the genotype of GG type as subsequent breeding materials.
The invention provides a method for breeding a wide-fruit papaya variety in an auxiliary manner based on the SNP molecular marker, the primer or the kit, which comprises the following steps:
1) extracting the genome DNA of the material to be selected;
2) taking the genomic DNA obtained in the step 1) as a template, and carrying out PCR amplification by using the primer to obtain a PCR amplification product;
3) sequencing the PCR amplification product in the step 2), determining the genotype of the 197 th base of the sequencing sequence, and when the genotype is GG, indicating that the papaya fruits of the material to be bred have the character of wide fruit shape, and using the papaya fruits as breeding materials for subsequent cross breeding.
The invention extracts the genome DNA of the material to be selected.
The invention is obtained by taking 340 parts of papaya collected from Mexico, south Africa, Guangxi China, Hainan China and the like as research materials and carrying out GWAS analysis and screening when screening SNP molecular markers. Therefore, the SNP molecular marker is obtained by screening based on a plurality of papaya varieties existing in the world. Therefore, the method provided by the invention is suitable for all papaya varieties.
The present invention is not particularly limited to the extraction of genomic DNA from a material to be cultivated, and a method for extracting plant DNA known in the art, such as a kit method or a CTAB method, may be used. In the embodiment of the invention, the genome DNA of the material to be bred is extracted by a kit method. The kit is a Tiangen plant genome DNA extraction kit, and is purchased from Tiangen corporation. After extracting the genome DNA, respectively detecting the content and the integrity of the DNA by using an ultramicro spectrophotometer and agarose gel electrophoresis. The verification shows that the electrophoresis strip is clear, no obvious protein residue exists in the gel hole, and OD 260 /OD 280 Between 1.8 and 1.9, the concentration is greater than>The DNA sample at 100 ng/. mu.L was subjected to the subsequent detection.
After the detection is qualified, the invention takes the genome DNA as a template and uses the primer to carry out PCR amplification to obtain a PCR amplification product.
In the invention, the reaction system of the PCR amplification is as follows:
2× Rapid Taq Master Mix 12.5μl
10 μ M Forward primer 1 μ l
10 μ M reverse primer 1 μ l
ddH 2 O 9.5μl。
The reaction program of the PCR amplification is 95 ℃ for 5 min; 30s at 95 ℃, 30s at 54 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃. The apparatus used in the PCR amplification of the present invention is not particularly limited, and a PCR apparatus known in the art may be used. After the PCR amplification is finished, preferably, the obtained PCR amplification product is purified, and then the 497 bp-long amplification fragment is collected for sequencing.
After obtaining the purified PCR amplification product, sequencing the PCR amplification product to determine the genotype of the 197 th base of a sequencing sequence, and when the genotype is GG, showing that the papaya fruits of the material to be bred have the character of wide fruit shape, and using the papaya fruits as breeding materials for subsequent cross breeding.
In the present invention, the sequencing was performed by Huada Gene Co. The sequencing is preferably bidirectional sequencing. And obtaining the sequence of the amplified product by splicing after obtaining the sequencing result. When the 197-bit base of the sequence is only G, the selection of the material to be bred is GG genotype, and the GG genotype indicates linkage with wider papaya fruit characters. When the material to be selected is AA genotype, the AA genotype is shown to be linked with narrower papaya fruit characters. Wherein the width of the narrow papaya fruits is preferably 67.7-75.6 mm.
The invention provides an SNP molecular marker Cpa03g017160:6929 related to the width of papaya fruits, which is beneficial to quickly detecting a papaya breeding material with high fruit width and assisting in cross breeding.
The SNP molecular marker related to the width trait of papaya fruit, the amplification primer, the detection kit and the application thereof provided by the present invention are described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Papaya whole genome SNP marker development
340 parts of papaya collected from Mexico, south Africa, Guangxi China, Hainan China and the like are sown in Wenchang base of Chinese tropical agricultural science institute, soil fertility is moderate, diseases and insect pests do not exist, the papaya is planted for many years at multiple points, phenotypic character data are collected, and the phenotypic character data are processed by Excel2016 and then used for subsequent analysis.
Taking 1-2 g of fresh tender leaves from a papaya plant, grinding by liquid nitrogen, and extracting papaya material DNA according to a selected radix asparagi plant genome DNA extraction kit (DP 305). Detecting DNA sample quality and concentration by using ultramicro spectrophotometer and 1% agarose gel electrophoresis, clear electrophoresis band, no obvious protein residue in gel hole, and OD 260 /OD 280 Between 1.8 and 1.9, the concentration is greater than>DNA samples at 100 ng/. mu.L were subjected to library sequencing.
The DNA is randomly broken into fragments of about 300bp by adopting an ultrasonic wave breaking (or enzyme digestion) method, and the construction of a sequencing library is completed by repairing the tail end of the DNA fragment, adding A to the 3' end, adding a sequencing joint pair, purifying and PCR amplifying. The library is qualified by quality inspection and then sequenced by an illumina platform. After the sequencing data are downloaded, the quality control of original data (Raw reads) is required according to a certain standard, and the filtering standard is as follows: (1) removing sequences with a linker (adapter), (2) removing a pair of sequences with a single-ended sequence nitrogen content >10%, and (3) removing a pair of sequences with a low-mass base number exceeding 50%. And removing the low-quality sequence, the linker sequence and the inaccurate sequence to carry out the next sequence alignment. Selecting a newly assembled papaya fruit material Kamiya genome from the subject group by referring to the genome, comparing Clean reads with a reference genome sequence by using BWA-mem software, sequencing results by using samtools software, removing PCR repetitive sequences by using GATK4.0 software, performing mutation detection, filtering a mutation set by using hard standards of QD > 2.0, QUAL > 30.0, FS < 60.0 and MQ > 40.0, and reserving a mutation site data set with statistical significance. And (4) filtering the mutation sites again by taking MAF (minor allele frequency) > = 0.05 and miss (deletion rate) < =0.2 as standards to obtain a high-quality mutation site collection.
GWAS analysis of papaya fruit width-related SNP sites
And annotating the variation sites by using the genome DNA interval feature description file, and respectively counting the number of the variation sites which fall in a gene coding region, a non-coding region, an intergenic region, non-synonymous mutation and the like. After obtaining a population genetic relationship matrix through population structure analysis, genome-wide association analysis is carried out by combining phenotypic character data, and significant correlation with papaya fruit width is detected on Chr03 chromosome as shown in figure 1 and figure 2 (-log 10: (a)P-value）>6) And a linkage disequilibrium region with the size of 20kb, a candidate gene Cpa03g017160 related to the size of the papaya fruit cavity is determined.
Screening candidate markers
The results of comparison of allele frequencies, differences in traits, and differences in gene expression levels based on SNP markers in the regions correlated with GWAS analysis are shown in table 1. SNP sites which are obviously related to the width of papaya fruits are screened in the Cpa03G017160 gene CDs region (Cpa 03G017160:6929 (A/G)).
TABLE 1 number of individuals with different genotypes for molecular markers and mean fruit Width (mm) in the population
Example 2
A pair of primers is designed by using Primer 5.0 software by taking a sequence containing SNP sites (Cpa 03G017160:6929 (A/G)) which are remarkably related to the width of the papaya fruit and are obtained by screening in example 1 as a template, wherein the sequences of the primers are as follows:
a forward primer: TGCTACTTCA GAAACATTAG (SEQ ID NO: 2);
reverse primer: GGAGACATCTTTCTTTATCTA (SEQ ID NO: 3).
The genome DNA of the material to be screened is subjected to common PCR amplification by using the primer pair, and the amplification system is as follows: 2 × Rapid Taq Master Mix 12.5 μ l, 10 μ M Forward primer 1 μ l, 10 μ M reverse primer 1 μ l, DNA template 1 μ l, ddH 2 O9.5. mu.l. The reaction procedure of PCR amplification is 95 ℃ for 5 min; 30s at 95 ℃, 30s at 54 ℃, 30s at 72 ℃ and 35 cycles; and 5min at 72 ℃ to obtain a 497bp DNA fragment, sequencing, wherein the sequencing result is similar to the sequence of the papaya related gene fragment SEQ ID NO:1, comparing and analyzing, and detecting the genotype carried by the SNP locus at the 197 th site of the sequence. Therefore, the method can detect or predict the width of the papaya variety fruits, further effectively select the width of the papaya variety fruits and accelerate the process of breeding the papaya variety with high fruit width.
Example 3
In order to verify the practicability of the SNP marker, 50 papayas (excluding 340 papayas used for development of the SNP marker) are randomly selected from a papayas growing area of Changchang base of the institute of biotechnology of Chinese tropical agrology academy of sciences, and then subjected to sequencing, genotyping and papaya fruit width property survey.
Table 250 papaya strains at Cpa03g017160:6929 different genotypes and fruit widths
Statistics of different genotypes and fruit widths of Table 350 papaya strains at Cpa03g017160:6929
As can be seen from Table 3, the Cpa03G017160:6929 locus G allele is very significantly associated with papaya fruit width.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> SNP molecular marker related to papaya fruit width character, amplification primer, detection kit and application thereof
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tgctacttca gaaacattag aaaaagaaga tcatacacat tagaggagaa tttgtttctc 60
ttttttgaca gtttatattt tcggtttgtt cttttgtttt tttttacttc attttgctct 120
cacaattact atcagatact ggaatgtcga cttgtgggaa aatctcttca caaagctgct 180
taacagtagc catggaratg atgacacaaa attgctgcgg agtttgaggg agtcctttca 240
gtggtacatc catgataacc ctgagctctt aaagaaacta aagcaattat tggcaaagca 300
aagggcgtct ttgtgttctt cttagtgtat attatatcat ttcttcagaa tccaccctaa 360
aagtgaatat aaataatttt aggaagtaaa tatttttttt ttgaaatcag tctgttgtcc 420
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Claims (5)
1. The application of a primer or a kit for amplifying SNP molecular markers related to the width character of papaya fruits in detecting or predicting the width of papaya fruits or in papaya breeding is provided, wherein the genotype of the papaya fruits is GG type, which indicates that the papaya fruits have wider characters;
selecting papaya materials with GG genotypes as follow-up breeding materials;
the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, wherein the 197 th site is A/G;
the primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 2 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3;
the kit comprises the primer.
2. A method for assisting in breeding wide-fruit papaya varieties by using primers for detecting papaya fruit width based on SNP molecular markers or a kit containing the primers is characterized by comprising the following steps:
1) extracting genome DNA of a material to be selected;
2) taking the genomic DNA obtained in the step 1) as a template, and carrying out PCR amplification by using the primer to obtain a PCR amplification product;
3) sequencing the PCR amplification product in the step 2), determining the genotype of the 197 th base of a sequencing sequence, and when the genotype is GG, indicating that the papaya fruits of the material to be bred have the character of wide fruit shape, and using the papaya fruits as breeding materials for subsequent cross breeding;
the primer comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 2 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3;
the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, wherein the 197 th site is A/G.
3. The method of claim 2, wherein the reaction system of the PCR amplification in step 2) is
2× Rapid Taq Master Mix 12.5μl
10 μ M Forward primer 1 μ l
10 μ M reverse primer 1 μ l
DNA template 1. mu.l
ddH 2 O 9.5μl。
4. The method of claim 2, wherein the reaction procedure of the PCR amplification in step 2) is 95 ℃ for 5 min; 30s at 95 ℃, 30s at 54 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃.
5. The method according to claim 2, wherein the amplified fragment of 497bp in length is collected for sequencing after the PCR amplification product is purified in step 2).
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| CN116426678B (en) * | 2023-03-27 | 2024-03-22 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to fructose content of papaya pulp and application thereof |
| CN116622876B (en) * | 2023-03-27 | 2024-03-22 | 中国热带农业科学院三亚研究院 | Haplotype molecular marker related to vitamin C content of papaya pulp and application thereof |
| CN117144055B (en) * | 2023-10-31 | 2024-03-22 | 中国热带农业科学院三亚研究院 | Application of haplotype molecular marker related to regulation and control of papaya fruit length |
| CN117512202B (en) * | 2024-01-04 | 2024-03-22 | 中国热带农业科学院三亚研究院 | SNP molecular marker remarkably related to papaya fruit chamber diameter, method and application |
| CN117512203B (en) * | 2024-01-04 | 2024-03-22 | 中国热带农业科学院三亚研究院 | Molecular marker related to papaya fruit width, method and application |
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| CN109486928A (en) * | 2018-12-28 | 2019-03-19 | 中国热带农业科学院热带生物技术研究所 | A kind of quickly early stage identification papaya method for distinguishing |
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| US10729082B2 (en) * | 2017-10-16 | 2020-08-04 | Benchbio Pvt. Ltd. | Papaya with novel traits and methods for producing such papaya plants |
| TWI721708B (en) * | 2019-12-12 | 2021-03-11 | 國立屏東科技大學 | A molecular marker related to papaya fruiting |
| CN112094937B (en) * | 2020-09-27 | 2022-05-17 | 中国农业科学院油料作物研究所 | SNP molecular marker related to pod and seed size on peanut A06 chromosome and application thereof |
| CN113846178B (en) * | 2021-10-15 | 2022-12-06 | 上海市农业科学院 | SNP molecular marker closely linked with major QTL of sweet corn grain size and application thereof |
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